Amplification method for solid phase immunoassays

ABSTRACT

The present invention is directed to immunoassays for detecting one or more target analytes in a fluid sample wherein the detection reaction occurs on a solid support and involves an amplification system. In particular, the invention is directed to making and using a test device having at least one site for detecting the presence of at least one target analyte, wherein a conjugate comprising dextran-polystreptavidin is immobilized at the test site(s) as a capture reagent for a complex containing the target analyte.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority from U.S. Provisional PatentApplication Ser. No. 60/604,378 filed on Aug. 25, 2004, which isexpressly incorporated by reference herein.

FIELD OF THE INVENTION

The present invention is directed to immunoassays for detecting one ormore target analytes in a fluid sample wherein the detection reactionoccurs on a solid support.

BACKGROUND OF THE INVENTION

Immunoassays have become methods of choice for rapid in vitrodiagnostics due in part to the ease of use, speed of reaction, andrelatively low cost. A common immunoassay format comprises a “capture”reagent immobilized on a solid substrate wherein the capture reagent iscapable of binding the target analyte, or a complex containing thetarget analyte, or a molecule that competes with the target analyte forbinding sites on the capture reagent. By localizing the capture reagentin a restricted zone or area, the result of the immunoassay is readilyvisualized or detected.

In particular, immunoassays in the form of lateral flow test strips forthe rapid analysis of fluids, including, but not limited to, body fluidssuch as urine, saliva, serum, plasma, whole blood, spinal fluid,amniotic fluid, and the like, or liquid extracts of specimens such asfeces, sputum, nasal swabs, etc., are popular as a means of determiningthe presence or absence of target analytes, such as hormones, drugs,allergens, and disease-related antigens. Examples of lateral flowdevices are disclosed in U.S. Pat. Nos. 4,094,647, 4,235,601, and4,361,537 to Deutsch et al., as well as U.S. Pat. No. 5,120,643 to Chinget al., U.S. Pat. No. 5,602,040 to May et al., U.S. Pat. No. 5,714,389to Charlton et al., and U.S. Pat. No. 6,352,862 to Davis et al., amongmany others.

Regarding ease of use, typically very little, if any, specimenprocessing is required prior to adding a liquid sample to a test strip.Therefore, preferably, the sensitivity of a lateral flow test device isadequate to detect or measure a minimal biologically and/orphysiologically significant concentration of particular target analyte,as may be expected to be contained within a fluid specimen as collected,without requiring additional processing to concentrate the targetanalyte prior to testing. Such restrictions on processing present achallenge when the biologically or physiologically meaningfulconcentration of a target analyte is low. Also, throat or nasal swabs,sputum, semen, vaginal and cervical secretions and other sorts ofviscous specimens usually require addition of a reagent to extract,breakdown or “thin” the sample to achieve an appropriately liquid stateto flow through a test device. Addition of such reagents leads tofurther dilution of the concentration of target analytes, compoundingthe problem of detecting low levels of target analytes that may bepresent in a mucus specimen, throat swab, semen, and the like.

Methods of increasing the amount of detectable label captured at a testsite have been described. U.S. Pat. No. 5,141,850 to Cole et al.,expressly incorporated by reference herein in its entirety, teaches thebinding of a “capturable” reagent to the target analyte, along withbinding of a labeled reagent, and the immobilization of a capturereagent specific for the capturable component. A preferred embodimentuses streptavidin conjugated to latex particles and localized within thepores of the porous carrier material at the test site by depositing theparticles under mild vacuum conditions. However, conjugation ofstreptavidin to latex particles adds to the labor and cost ofmanufacturing the test device and entrapping particles within the poresof the carrier material is not equivalent to chemically or physicallybinding, directly or indirectly, the capture reagent to the carriermaterial. Moreover, entrapment of latex particles within a carriermaterial requires porosity sufficient to contain particles, but is notdesirable for use in connection with samples containing cellularcontaminants and/or debris, such as sputum samples, semen, vagina andcervical secretions, whole blood, and the like.

Another approach to amplification of detection within an immunoassay isto use polymeric carriers in preparing various conjugated reagents. Suchpolymeric conjugates are disclosed in U.S. Pat. No. 5,543,332 to Lihmeet al. (expressly incorporated by reference herein in its entirety). Oneexample is a dextran-polystreptavidin conjugate. Conjugation of multiplemolecules of streptavidin to each dextran molecule significantlyincreases the number of available streptavidin molecules within alocalized area for binding to a receptor molecule, namely biotin,thereby amplifying the amount of biotinylated molecules captured withina localized area.

It is known to use dextran-polystreptavidin for preparing conjugatereagents that react in a “free” form (not immobilized on a solidsupport) in solution, for binding to or competing with a target analyte.Such reagents are taught in U.S. Pat. No. 5,543,332 in connection withELISA and “dot-blot” immunoassays wherein biotin is immobilized on asolid support and, thus, biotin serves as the capture reagent. Morerecently, polymeric conjugates are described in U.S. Pat. No. 6,709,611to Lassen et al. in connection with an assay for detecting respiratorysyncytial virus (RSV), wherein a dextran polymeric carrier is used as a“mobile solid phase”, for example, to prepare labeling reagents.

SUMMARY OF THE INVENTION

The present invention is directed to immunoassays incorporating anamplification system. In particular, the invention is directed to makingand using a test device having at least one site for detecting thepresence of at least one target analyte, wherein a conjugate comprisingdextran-polystreptavidin is immobilized at the test site(s) as a capturereagent. Applicants discovered that dextran-polystreptavidin can beimmobilized on a porous carrier material, such as nitrocellulose, byaltering the “curing” conditions compared to those typically used toimmobilize antibodies, antigens, or other proteins that comprisestandard capture reagents. The immobilized dextran-polystreptavidinsubstantially remains as a discrete band.

Streptavidin moieties within the immobilized dextran-polystreptavidinconjugate bind specifically to biotin; thus a biotinylated component,such as a biotinylated binding partner to the analyte, is used. Forexample, biotinylated antibodies specific for a target analyte can beutilized, or if the target analyte is itself an antibody, then abiotinylated antigen can be used. Likewise, hormones, receptors, and thelike can serve as target analytes, and appropriate ligands thatspecifically bind to the hormones, receptors, or other target analyteare conjugated to biotin. In one embodiment, biotinylated conjugates aredried or lyophilized in a separate area from other dried components,such as dried labeled conjugates, because separating the biotinylatedconjugate in a separate site from the site containing a labeledconjugate appears to further improve the sensitivity of the device usingdextran-polystreptavidin as a capture component. In one embodiment, thebiotinylated conjugate, such as a biotinylated antibody or antibodyfragment capable of specifically binding to a target analyte, is placedupstream of the labeled component, which is, in turn, upstream from thetest site(s).

In one embodiment, a biotinylated antibody capable of specificallybinding the target analyte is dried within a piece of porous plastic,such as product #4588 from Porex (Fairburn, Ga.). The labeled component,for example, another antibody capable of binding the target analyteconjugated to a detectable label, including but not limited to an enzymeor enzyme fragment, metal sol, latex particle, fluorescent molecules ordye, is dried within a glass fiber pad, for example, glass fiber #66078from Gelman Sciences (Ann Arbor, Mich.), downstream from thebiotinylated conjugate. Continuing downstream, thedextran-polystreptavidin capture component, such as product #OA222commercially available from DakoCytomation (Carpinteria, Calif.), isdried on a porous material, such as Millipore HiFlow Plus HF13502 orHF12002 (Millipore, Billerica, Mass.), although other porouschromatographic materials, such as those made from nitrocellulose,paper, or nylon may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an expanded perspective of one embodiment of a test stripfor performing a lateral flow immunoassay comprising two separate porouscarrier materials.

FIG. 2 is an expanded perspective of another embodiment of a test stripfor performing a lateral flow immunoassay comprising three separatecomponents.

FIG. 3 illustrates an expanded perspective of a composite test stripcomprising five components and a backing support material.

FIG. 4 shows a housing containing two separate test strips for twodifferent antigens.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows one embodiment of the invention in a lateral flow format. Afirst porous material 10, such as nitrocellulose or other suitableporous substrate known in the art, contains at least one test site 12comprising immobilized dextran-polystreptavidin, for example, product#OA222 commercially available from DakoCytomation (Carpinteria, Calif.).Upstream of the first porous material 10 is a second porous material 14that serves as the sample application site 16 and as the conjugate site18 containing dried biotinylated antibodies capable of specificallybinding a target analyte and also containing antibodies conjugated to adetectable moiety (label) and capable of specifically binding a targetanalyte. In one embodiment, the second porous material 14 overlaps thefirst porous material 10 or otherwise forms contact that permits lateralflow of the fluid specimen from the first porous material 10 into thesecond porous material 14. An optional control site 13 is located on thefirst porous material 10 downstream of the test site(s) 12. The porousmaterial may be beaded agarose, beaded polyacrylamide, porous glass,cellulose or other materials permeable to liquid and compatible with theassay components and test analyte.

FIG. 2 shows another embodiment of the invention wherein the sampleapplication site 16 is contained in a third porous material serving as areceiving area 20 separate from the conjugate site 18. Such systems areknown in the art. In one embodiment, the material forming the receivingarea 20 overlaps the second porous material 14 containing the conjugatesite 18 or otherwise forms contact that permits lateral flow of thefluid specimen.

Yet another embodiment of the invention is shown in FIG. 3. The thirdporous material serving as the receiving area 20 further contains adried biotinylated conjugate and is referred to as the biotinylatedconjugate pad 22. The biotinylated conjugate pad 22 is shown upstreamof, and in fluid contact with, a second porous material 14 containingthe dried labeled conjugate, but the order may be reversed so that thebiotinylated conjugate is downstream from the labeled conjugate. Furtherdownstream from both conjugates, is the first porous material 10containing at least one test site 12 comprising immobilizeddextran-polystreptavidin conjugate.

As further shown in FIG. 3, with certain types of specimens, additionalfiltering is beneficial to reduce streaking and other backgroundproblems that may interfere with interpretation of test results.Additional filtering is provided by including a blank piece 21 of porousmaterial, such as a piece of glass fiber, between the biotinylatedconjugate pad 22 and the first porous material 10 which, in oneembodiment, is directly between the biotinylated conjugate pad 22 andthe second porous material 14 containing the dried labeled conjugate.

In one embodiment, the device also includes an absorbent pad 24 at thedistal end of the first porous material 10 to function as a “sink” forthe fluid specimen and facilitate adequate capillary flow through thetest device. The absorbent pad 24 is comprised of paper, such as D28from Whatman (Fairfield, N.J.) or other suitable absorbent material suchas cotton, cellulose and other materials as known in the art. In oneembodiment, all components are held in place via a backing support 26,such as vinyl with adhesive on one side, to maintain appropriatefunctional relationships and ensure proper performance.

As a specific example of one embodiment, a lateral flow test device fordetecting influenza A and influenza B antigens is described in detail inthe examples below.

EXAMPLE 1

Biotinylated Antibodies

An antibody specific to influenza A antigens, for example monoclonalantibody product #1G4 from Diagnostic Products Corporation (DPC; LosAngeles Calif.) and an antibody specific to influenza B antigens, suchas monoclonal antibody product #B35G (DakoCytomation, CarpinteriaCalif.) and/or monoclonal antibody product 8F10E10 (Argene Inc., NorthMassapequa N.Y.), were separately coupled to biotin (for example,Sulfo-NHS-LC biotin, Pierce Biotechnology, Inc., Rockford Ill.) bymethods known in the art. In one embodiment, coupling was done at aconcentration of 2 mg of antibody per ml and the final conjugatedproduct was diluted approximately 1:75 for use in making the testdevice.

In one embodiment, biotinylated antibodies were vacuum dried by methodsknown in the art within a piece of porous plastic, for example, Porex#4588, which is cut into strips for the test device. However, otherporous material, such as glass fiber, can be used. In one embodiment,the biotinylated antibodies were dried into separate pieces of porousmaterial. In another embodiment, the biotinylated antibodies forinfluenza A and for influenza B were dried within the same porousmaterial. The dried antibodies must be capable of becoming rehydratedand soluble in the liquid specimen as it moves through the device.

EXAMPLE 2

Labeled Antibodies

Antibodies specific for influenza A antigens and antibodies specific forinfluenza B antigens were conjugated with detectable labeling moieties.Either distinguishable labels or the same label can be used to labeleach different antibody. Suitable labels include, but are not limitedto, metal sols, latex particles, enzymes, fluorescent molecules, and thelike, as known in the art. One label is carboxylate-modified latexparticles (Seradyn, Indianapolis Ind.) available in various colors.Antibodies were coupled to carboxylate-modified latex particles usingcarbodiimide N-hydroxysuccinimide linkage, by standard procedures knownin the art. Separate monoclonal antibody products from those used toprepare biotinylated reagents can be used, if available, or the sameantibody product can be used for biotin-conjugation and for conjugationwith a labeling moiety.

The labeled antibodies were vacuum dried by methods known in the artwithin a porous material, such as glass fiber, and the porous materialwas cut into sections for use in a test device. The labeled antibodiescan be dried together or each can be dried in separate pieces of porousmaterial. The labeled antibodies must be capable of being rehydrated andsoluble in the liquid specimen as it moves through the device.

EXAMPLE 3

Test Strip with Test Site and Optional Control Site

Dextran-polystreptavidin was immobilized as a capture reagent at a testsite on a porous material. In one embodiment, the porous material wasnitrocellulose, such as Millipore HiFlow Plus HF12002 or HF13502(Millipore, Billerica Mass.). Dextran-polystreptavidin reagent was mixedin phosphate buffed saline (PBS) supplemented with sucrose(approximately 2.5%) and EDTA (approximately 0.33 mM), and deposited inone or more discrete bands or zones on the porous material, and allowedto dry or “cure” for about 10 to 15 hours,at 60° C. or about 7 to 14days at room temperature. Although these time and temperature conditionswere sufficient to immobilize the dextran-polystreptavidin conjugate onthe porous material, they are exemplary and the invention is not limitedto these specific conditions, but includes other conditions that resultin immobilization

A simple method for checking the quality of immobilization was to add afluorescent molecule, such as fluorescein, to the PBS striping solutioncontaining the dextran-polystreptavidin conjugate. After the stripingprocess and curing period, a sample test strip was wetted at one end topermit an aqueous solution to flow laterally through the test site andthe integrity of the striped band was checked or monitored byobservation under ultraviolet light to ensure the test band remaineddiscrete. Such methods are known and have become standard in the art.

To serve as a control site, antibodies specific to mouse IgG, such asgoat-anti-mouse or rabbit-anti-mouse antiserum, were deposited at adiscrete band or zone on the porous material and allowed to dry at 60°C. for about 45 minutes to about one hour or at room temperature forabout 10-15 hours.

EXAMPLE 4

Assembling the Composite Strip

As shown in FIG. 3, various components comprising the composite stripwere anchored or secured on a backing support 26, such as an adhesivebacking material, for example, white vinyl having a thickness of about0.001 inches to about 0.03 inches. Such material is available fromnumerous sources. The first porous material 10 of the composite teststrip, containing at least one test site 12, and optionally containing acontrol site 13, was positioned downstream of a conjugate pad 14containing the labeled conjugate. The labeled conjugate pad 14 mustoverlap or make contact with the test strip 10 such that fluid possiblycontaining a target analyte moves laterally through the labeledconjugate pad 14 and through the test site 12. The use of an absorbentmaterial 24 at the far downstream end of the first porous material 10facilitated wicking or capillary flow through the test site 12 and actedas a sink to contain the fluid sample. Optionally, a blank filter 21(free from immunoreactive reagents) was placed between the biotinylatedconjugate pad 22 and the second porous material 14 containing thelabeled conjugate to reduce streaking and other background problems thatare associated with specimens containing high levels of cellularcomponents and debris.

EXAMPLE 5

Device for Detecting Influenza A and B Antigens

FIG. 4 illustrates a single device to detect two or more differenttarget analytes, such as influenza antigens A and B, containing twoseparate composite test strips housed within a single housing orcassette 28. Each test strip has a test site 12 comprising immobilizeddextran-polystreptavidin and a control site 13 with immobilizedanti-mouse IgG antibodies although other capture reagent suitable forcontrol purposes may be used. It will be appreciated that the twoseparate composite test strips may detect the same analytes.

Each of the test devices within the cassette 28 comprised the compositecarrier membrane shown in expanded perspective in FIG. 3. In oneembodiment, separate biotinylated conjugate pads 22 were used, upstreamof the labeled conjugate pad 14, with one comprising dried biotinylatedantibodies directed against influenza A antigens, and the othercomprising dried biotinylated antibodies to influenza B antigens. In oneembodiment, a blank piece 21 of glass fiber or other filtering materialwas placed directly between each biotinylated conjugate pad 22 andadjacent labeled conjugate pad 14.

EXAMPLE 6

Using a Test Device to Detect Influenza A and/or B Antigens

Acceptable specimens for use with an influenza test device includednasal washes, nasal swabs, throat swabs, sputum and the like. Samplescould be tested immediately upon collection or, alternatively, samplescould be stored in suitable transport and storage medium, for exampleM4® (Remel, Lenexa Kans.) at 2-8° C. for up to about 72 hours prior totesting.

Freshly collected specimens could be tested immediately without the useof transport medium. Alternatively, specimens could be placed into asuitable transport medium, maintained at 2-8° C. and tested within 72hours after collection. Frozen specimens in a suitable liquid viraltransport medium stored at −20° C. or below in a non-defrosting freezercould be tested up to six months after collection. Multiple freeze-thawcycles should be avoided.

The following transport media have been evaluated and found to becompatible: Amies Medium; Bartels Vital Transport Medium; Cary BlairMedium; Earle's Minimum Essential Medium (EMEM); EMEM with 1% BovineSerum Albumin; EMEM with 1% Lactalbumin hydrolysate; Hank's BalancedSalt Solution; Liquid Stuarts Medium; M4™; M4-RT™; M5™; PhosphateBuffered Saline (PBS); PBS with 0.5% Bovine Serum Albumin; PBS with 0.5%Gelatin; Saline (normal); Sucrose Phosphate; Tryptic Soy Broth with 0.5%Bovine Serum Albumin; Tryptic Soy Broth with 0.5% gelatin; VealInfuision Broth; and Veal Infusion Broth with 0.5% Bovine Serum Albumin.

A foil pouch may contain one single-use test device with two membranestrips. In one embodiment, the strips in the device contain,respectively, antibodies to influenza A or B. A specimen diluent may beincluded; the specimen diluent may be a buffered saline solution withdetergent, a mucolytic agent, and preservative. In one embodiment,flexible plastic tubes for specimen preparation and disposable transferpipettes are included; the pipettes with marked gradations atapproximately 0.1 ml increments. A Flu A+/B− control swab, which is adry swab containing inactivated influenza A antigen, and a Flu B+/A−control swab, which is a dry swab containing inactivated influenza Bantigen, may be included.

The test device is removed from the foil pouch when the assay is to beperformed. It is placed on a flat surface. If the kit and/or componentshave been refrigerated, they are allowed to equilibrate to roomtemperature.

For swab specimens without dilution (swab not submerged in a sufficientvolume of transport media to allow processing of 0.1 ml) in transportmedia (including quality control swabs provided with the kit), 25 drops(approximately 0.6 ml) of specimen diluent are dispensed into a dilutiontube. The swab specimen is placed in the tube and the tube is mixedthoroughly or vortexed to release bound antigenic material from theswab. The swab is rotated firmly against the tube walls then squeezedagainst the sides of the tube during removal.

For all specimens except swab specimens without transport media, 5 drops(approximately 0.1 ml) of specimen diluent is dispensed into thedilution tube. The specimens are mixed well and 0.1 ml (first moldedgraduated mark from the tip of transfer pipette) of liquid specimen(nasal wash or specimens in transport medium) is transferred into adilution tube.

Using a transfer pipette, 0.2 ml (second graduated mark from tip ofpipette) of specimen is dispensed into the center of the sample well ofthe test device. Test results are visually read after 15 minutes or upto 30 minutes as will be subsequently described. A strong positiveresult may be apparent sooner than 15 minutes.

Specimens, if needed, are exposed to a suitable extraction solution. Forexample, an aqueous solution comprising about 3% bovine serum albumin,about 3% non-ionic detergent, 0.1% casein, 0.1% N-acetyl-cysteine, 0.5 Msodium chloride, 25 mM monobasic sodium phosphate, 75 mM dibasic sodiumphosphate, 0.05% Proclin 300, adjusted to a pH of about 7.4, is aneffective extraction solution. For fresh swab specimens (not in atransport medium), the swab is placed into approximately 0.6 ml ofextraction solution in a flexible plastic tube. The specimen is mixed,preferably via vortexing, to release bound potentially antigenicmaterial, and the swab is squeezed by compressing the walls of theflexible tube while removing the swab. For liquid specimens, includingnasal washes or transport media exposed to swab specimens, about 0.1 mlof extraction solution is mixed with about 0.1 ml of specimen.

About 0.20 to 0.25 ml of the extract is transferred to the sampleapplication area of the test device. The liquid specimen moves laterallyvia wicking or capillarity through the device, rehydrating the driedbiotinylated conjugates and the dried labeled conjugates. If the targetanalyte is present, it becomes sandwiched between the biotin-conjugateand the labeled conjugate. The streptavidin moieties of thedextran-polystreptavidin capture component bind the biotinylatedconjugate regardless of whether the biotinylated conjugate is also boundto target analyte. However, if complexes of the biotinylated antibodies,target analyte and, hence, the labeled conjugate have formed, thelabeled conjugate will form a detectable region at the capture site asthe biotinylated conjugate is captured by immobilized streptavidinmoieties. Optionally, excess unbound labeled conjugate is captured at acontrol site downstream of the test site(s).

Test results are interpreted as follows. The test device has twoseparate read windows; the one on the left for Flu A and the one on theright for Flu B as depicted in FIG. 4. A positive test, indicating thatantigen is present in the specimen, is indicated by two black-coloredbands; one in the test (T) region and one in the control (C) region. Anegative test, indicating that antigen was not detected, is indicated byonly one black-colored band in the control (C) region. An invalid testresults in no black-colored band in the control (C) region. A complete,black, clearly visible test line of any intensity should be interpretedas positive. Invalid results due to excessively mucoid specimens may berepeated using twice the normal volume of specimen diluent during thedilution step.

Results may be reported as follows: positive for influenza A and/orinfluenza B antigen; negative for influenza A and/or influenza Bantigen; or infection due to influenza A or B cannot be ruled out sincethe antigen present in the specimen may be below the detection limit ofthe test. Specimens in which a negative result was obtained should becultured for confirmation.

For internal quality control, a procedural control may be included inthe test. A colored band appearing on the control band (C) region isconsidered an internal positive procedural control, indicating properperformance and reactive reagents. A clear background in the resultsarea is considered an internal negative control. If the test has beenperformed correctly and reagents are working properly, the backgroundwill clear to give a discernible result.

For external quality control, positive and negative controls should beassayed with each new test kit lot number, following state and localrequirements.

Quality control swabs that are Flu A+/B− and Flu A−/B+ are provided withthe kit. They should be processed in accordance with the procedure forswab specimens without transport media. If controls do not perform asexpected, patient results should not be reported.

Both viable and nonviable influenza A and B viruses were detectable withthe Xpect™ Flu A&B test. Due to low levels of virus shedding, inadequatespecimen collection, or improper handling or transport, a negative testresult does not rule out the presence of influenza virus. Consequently,the results from the Xpect™ Flu A&B test should be used in conjunctionwith other clinical findings to establish a diagnosis. A positive testdoes not rule out the possibility of co-infection with another pathogen.

In the United States, influenza is most prevalent during the wintermonths. During peak periods, up to 30% of specimens tested may beculture positive for influenza. The proportion of influenza A positivespecimens compared to influenza B can vary dramatically from year toyear, ranging from about 50% to 99%.

The performance of the Xpect™ Flu A&B test was evaluated at three siteslocated in the north, south and east regions of the United States. Theclinical trial sites included a children's hospital (pediatricpopulation), a university hospital (primarily adult population), and areference laboratory (adult and pediatric (60/40) population). For allspecimens evaluated, the overall sensitivity of the Xpect™ Flu A&B testwhen compared to culture was 92.2% (71/77) for influenza A and 97.8%(45/46) for influenza B. The overall specificity was 100% for bothinfluenza A (314/314) and influenza B (345/345). For influenza A, therewere six samples that were culture positive and Xpect™ Flu A&B negative.For influenza B, there was one sample that was culture positive andXpect™ Flu A&B negative. Four of five discrepant samples available foranalysis were positive by reverse transcription PCR (RT-PCR).

Nasal Wash (n=239)

Influenza A

-   92.5% Sensitivity (37/40); 95% CI=79.6-98.4%-   100% Specificity (199/199); 95% CI=98.2-100%    Influenza B-   100% Sensitivity (36/36); 95% CI=90.3-100%

100% Specificity (203/203); 95% CI=98.2-100% Culture Results OVERALLA+/B− A−/B+ A−/B− Xpect ™ Flu A+/B− 37  0 0 A&B Results A−/B+ 0 36 0A−/B−  3* 0 163*RT-PCR was performed on the three discrepant results. One of the threespecimens was negative by PCR, two were positive.

Test performance by individual site: Sensitivity Specificity Site # %95% CI # % 95% CI FLU A 1 0/0 NA NA 1/1 100 NA 2 3/5 60.0 14.7-94.769/69 100 94.8-100 3 34/35 97.1 85.1-99.9 129/129 100 97.2-100 FLU B 10/0 NA NA 1/1 100 NA 2 0/0 NA NA 74/74 100 95.1-100 3 36/36 10090.3-100  128/128 100 97.2-100Throat Swab (n=30)Influenza A

-   100% Sensitivity (10/10); 95% CI=69.2-100%-   100% Specificity (20/20); 95% CI=83.2-100%    Influenza B-   100% Sensitivity (4/4); 95% CK=39.8-100%

100% Specificity (26/26); 95% CI=86.8-100% Culture Results OVERALL A+/B−A−/B+ A−/B− Xpect ™ Flu A+/B− 10 0 0 A&B Results A−/B+ 0 4 0 A−/B− 0 016

Test performance by individual site: Sensitivity Specificity Site # %95% CI # % 95% CI FLU A 1 10/10 100 69.2-100 18/18 100 81.5-100 2 0/0 NANA 2/2 100 15.8-100 3 NA NA NA NA NA NA FLU B 1 4/4 100 39.8-100 24/24100 85.8-100 2 0/0 NA NA 2/2 100 15.8-100 3 NA NA NA NA NA NANasal Swab (n=122)Influenza A

-   88.9% Sensitivity (24/27); 95% CI=70.8-97.7%-   100% Specificity (95/95); 95% CI=96.2-100%    Influenza B-   83.3% Sensitivity (5/6); 95% CI=35.9-99.6%

100% Specificity (116/116); 95% CI=96.9-100% Culture Results OVERALLA+/B− A−/B+ A−/B− Xpect ™ Flu A+/B− 24 0 0 A&B Results A−/B+  0 5 0A−/B−  3*  1* 89*RT-PCR was performed on two of the four discrepant specimens that wereavailable (one influenza A and one influenza B). Both specimens werepositive by PCR.

Test performance by individual site: Activity Specificity Site # % 95%CI # % 95% CI FLU A 1 24/27 88.9 70.8-97.7 91/91 100 96.0-100 2 0/0 NANA 4/4 100 39.8-100 3 NA NA NA NA NA NA FLU B 1 5/6 83.3 35.9-99.6112/112 100 96.8-100 2 0/0 NA NA 4/4 100 39.8-100 3 NA NA NA NA NA NA

The analytical sensitivity was evaluated using twelve influenza strains;six influenza A and six influenza B. Each viral strain was quantitatedby CEID₅₀ determinations and titrated until a positive endpoint wasreached using the Xpect™ Flu A&B test. The amount of virus at theendpoint dilution, expressed as CEID₅₀ per test, was calculated as ameasure of analytical sensitivity. Detection Limit Influenza Strain TypeCEID₅₀ A/Puerto Rico/8/34 (H1N1) A 8.9 × 10³ A/Fort Monmouth/1/47 (H1N1)A 7.9 × 10¹ A/New Jersey/8/76 (H1N1) A 8.9 × 10¹ A/Hong Kong/8/68 (H3N2)A 2.8 × 10¹ A/Victoria/3/75 (H3N2) A 8.9 × 10² A/Port Chalmers/1/73(H3N2) A 4.0 × 10¹ B/Lee/40 B 7.9 × 10³ B/Allen/45 B 4 B/Maryland/1/59 B6 B/GL/1739/54 B 8.9 × 10¹ B/Taiwan/2/62 B 3 B/Hong Kong/5/72 B 1.58 ×10² 

Thirty-six microorganisms were evaluated with the Xpect™ Flu A&B test.No cross-reactivity was observed for influenza A or influenza B.Bacteria and yeast isolates were tested at 10⁸ colony-forming units perml concentration. Viral isolates were tested at concentrations of 10⁴ to10⁵ TCID₅ (tissue culture infectious dose) per ml concentration. Thefollowing organisms were tested in the Xpect™ Flu A&B test.Acinetobacter baumannii, Bordetella pertussis, Candida albicans,Enterococcus faecalis, Escherichia coli, Gardnerella vaginalis,Haemophilus influenzae, Klebsiella pneumoniae, Lactobacillus casei,Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis,Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca,Neisseria subflava, Proteus vulgaris, Pseudomonas aeruginosa, Serraliamarcescens, Staphylococcus aureus (Cowan), Staphylococcus epidermidis,Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenesGroup A, Streptococcus Group B, Streptococcus Group C, StreptococcusGroup F, Adenovirus Type 5, Coronavirus, Coxsackievirus B5,Cytomegalovirus, Parainfluenza (Sendal) Type 1, Parainfluenza Type 2,Parainfluenza Type 3, Respiratory Syncytial Virus A, Rhinovirus Type 14.

The following substances were tested with the Xpect™ Flu A&B test and nointerference was observed in the assay for any substance tested at theindicated levels: whole blood (2%), three mouthwashes (25%), threethroat drops (25%), three nasal sprays (25%), 4-acetomidophenol(acetaminophen) (10 mg/ml), acetylsalicylic acid (20 mg/ml),chlorpheniramine (5 mg/ml), dextromethorphan (10 mg/ml), diphenhydramine(5 mg/ml), guaiacol glyceryl ether (guaifenesin) (20 mg/ml),oxymetazoline (10 mg/ml), phenylephrine (25 mg/ml), phenylpropanolamine(20 mg/ml).

Reproducibility testing was conducted at four sites, including onein-house site, on four separate days with six blinded samples. Theliquid samples consisted of diluted influenza A and influenza B antigensintended to read weakly positive or negative with the Xpect™ Flu A&Btest. Ninety-nine percent of the 96 samples tested produced the expectedresult.

EXAMPLE 7

Cazacu et al. (J. Clinical Microbiology, 42:3661-3664, 2004) reportresults of specificity and sensitivity testing of one embodiment of theinvention directed to the detection of influenza A and B antigens ascompared to results obtained from viral culture. Overall, the observedspecificity was 100% for influenza A and B antigens in all specimentypes tested, while sensitivity was 100% in throat swabs, 96.1% in nasalwashes and 87.9% in nasopharyngeal swabs.

The test results obtained with the rapid lateral-flow chromatographicmembrane immunoassay (Xpect™ Flu A&B, Remel Inc., Lenexa Kans.) werecompared to those with the reference standard of viral culture in 400respiratory specimens collected from children and adults who presentedbetween January and April 2003 with respiratory or flu-like symptoms toone of three hospitals in three geographically distinct areas: TexasChildren's Hospital, Houston, Tex. (n=166), University of South FloridaAffiliated Hospitals and DSI Reference Laboratory, Fort Myers, Fla.(n=151), and SUNY Upstate Medical University Affiliated Hospitals, NewYork, N.Y. (n=83). Most (239 of 400, or 59.75%) specimens were nasalwashes, 122 of 400 (30.5%) were nasopharyngeal swabs, 30 of 400 (7.5%)were throat swabs, 4 of 400 (1%) were tracheal aspirates, 3 of 400(0.75%) were sputum, and 2 of 400 (0.5%) were obtained bybronchoalveolar lavage. Both fresh samples and samples cryopreserved forless than three months were analyzed at each institution.

Virology technicians or technologists at all centers receivedinstruction on the test procedures and were required to pass (>90%) ablinded proficiency test on six coded samples, administered daily forfour days, prior to starting testing on clinical samples included inthis study. Rapid tests were performed according to the manufacturer'sinstructions during weekday, day shift hours. Briefly, the test detectedinfluenza virus antigen in a test device that contained a sample well inwicking communication to reading wells that contained separate membranestrips for influenza A and influenza B viruses (FIG. 4). Each sample wasmixed with a specimen diluent that contained buffered saline, detergent,a mucolytic agent, and preservative. Then, 0.20 ml was transferred bypipette into the middle of the test well of the device. A positive testwas indicated by two black bands in the reading well, one in the test(T) region and one in the control (C) region. A negative test wasindicated by only one black band in the C region. The absence of anyblack bands in the T or C regions represented an invalid test. Testreadings were performed and recorded after 15 and 30 min incubation.Quality control procedures were performed and recorded for each test runor 24-h period and included both FluA+/FluB− and FluA−/FluB+ controlsprovided by the test kit, as well as in-house positive and negativecontrols for each virus. All specimens were also inoculated that sameday into cell culture monolayers of human foreskin fibroblast, humanlung carcinoma (A549), human epithelial (HEp2), and rhesus monkey kidney(RhMK) cells and examined daily for cytopathic effect using lightmicroscopy. Hemadsorption with a 0.4% suspension of guinea pig red bloodcells was performed on days 2, 5, and 14 of incubation of RhMK cellcultures. Virus identification was confirmed by an immunofluorescenceassay with type-specific antibodies. At one institution (TexasChildren's Hospital), all picornaviruses were discriminated by acidlability testing to distinguish between rhinoviruses and enteroviruses.Samples with discrepant results between viral culture and the rapidinfluenza virus test were cryopreserved and analyzed by RT-PCR usingprimers able to detect and differentiate influenza A and B viruses.

Viral cultures positive for influenza virus, type A or B, wereconsidered true positives. Sensitivity, specificity, and positive andnegative predictive values were calculated using two-by-two contingencytables. Differences between tests were analyzed using Fisher's exacttest. Because rapid testing for influenza virus may be performed toscreen persons during a pandemic or other event affecting large numbersof people, confidence intervals (CI) for proportions were calculated, toestimate with 95% confidence, the intervals that contain thesensitivity, specificity, and predictive values for the generalpopulation, estimated in this analysis to be 1,000,000. Using theconfidence intervals, results from the sample size of 400 weregeneralized to a large population, and the estimation predicted theinterval that contained the rapid assay's performance.

The importance of this test performance is relevant in view of concernsabout global pandemics of influenza, severe acute respiratorysyndrome-associated coronavirus, and the possibility of a biologicalwarfare event, indicating a need to screen large numbers of persons withfebrile respiratory illness of undetermined etiology.

Of 400 specimens, 207 (51.75%) had a negative viral culture and 193(48.25%) grew at least one virus, as shown in Table 1. TABLE 1 Virusesisolated from respiratory samples collected from three participatingcenters during the 2003 influenza season No. (%) of each Virus isolatedvirus isolated Influenza A virus  79 (40.1) Influenza B virus  46 (23.4)Respiratory Syncytial Virus  25 (12.7) Parainfluenza virus  12 (6.1)Adenovirus  8 (4.1) Picornavirus  7 (3.6) Rhinovirus  7 (3.6)Enterovirus  4 (2.0) Cytomegalovirus  7 (3.6) Herpes Simplex Virus  2(1.0) Total no. of viruses isolated 197**Four samples had dual viral infections, as described in the text.Dual viral infections were detected in four specimens (one withinfluenza A virus and adenovirus, one with influenza A virus andrespiratory syncytial virus, one with influenza B virus andcytomegalovirus, and one with picornavirus and adenovirus), to give atotal of 197 viruses isolated during the study period. The mean durationthat elapsed until viral cultures were detected as positive was4.43±2.87 days, and 87.6% of cultures were positive with seven days.

The overall sensitivity of the rapid test to detect both types ofinfluenza virus was slightly higher (95.2% versus 94.4%) at the 30-minreading than the 15-min reading, but specificity and predictive valueswere essentially the same at both readings. No significant differenceswere observed in the ability of the test to detect influenza A or Bvirus, and the results were generalized with 95% confidence to apopulation of at least 1,000,000, as shown in Table 2. TABLE 2Performance of the lateral-flow chromatographic membrane immunoassay(RA), at 15- and 30-min readings, compared to that of viral culture (CX)for detection of influenza A and B viruses^(a) RA+ and RA+ and RA− andRA− and RA CX+ CX− CX+ CX− Sensitivity(CI) Specificity CI) PVP (CI) PVN(CI) At 15 min Both A and B 118 0 7 275 94.4 (92.1-96.6) 100 (98.8-100)100 (98.8-100) 97.5 (95.9-99) A only 73 0 6 321 92.4 (89.8-95) 100(98.8-100) 100 (98.8-100) 98.2 (96.8-99.5) B only 45 0 1 354 97.8(96.4-99.2) 100 (98.8-100) 100 (98.8-100) 99.7 (98.7-100) At 30 min BothA and B 119 0 6 275 95.2 (93.1-97.3) 100 (98.8-100) 100 (98.8-100) 97.9(96.4-99.3) A only 74 0 5 321 93.7 (91.2-96.1) 100 (98.8-100) 100(98.8-100) 98.5 (97.2-99.7) B only 45 0 1 354 97.8 (96.4-99.2) 100(98.8-100) 100 (98.8-100) 99.7 (98.7-100)^(a)CI, 95% confidence interval results generalized to population of 1million;PVP, predictive value positive;PVN, predictive value negative;A, influenza type A virus;B, influenza type B virus.No significant differences were found when performance statistics wereanalyzed by center site.

No false-positive tests were observed at either the 15- or 30-minincubation time for the rapid test. There were seven false-negativeresults at 15 min, and six false-negative results at the 30-minincubation time. Only nasal washes (three) and nasopharyngeal swabs(three) had false-negative results. Overall, the specificity of therapid assay was 100% for all specimen types. Sensitivity was 100% inthroat swabs, 96.1% in nasal washes, and 87.9% in nasopharyngeal swabs.

False-negative results were detected at all three centers, and testingby RT-PCR of the five specimens with apparent discrepant resultsconfirmed four of the false-negative test results, as shown in Table 3.Spec- RA^(d) at: imen Spec- Influenza Days to 15 30 no. Site^(a)imen^(b) virus type CX+^(c) min min RT-PCR 16 DSI NP A 3 — — A+ 41 DSINP A 3 — Weak + NT^(e) 77 DSI NP A 2 — — NT^(f) 163 UMU NW A 3 — — A+171 UMU NW A^(g) 3 — — A−/B−^(g) 361 TCH NW A 5 — — A+ 118 DSI NP B 3 —— B+^(a)DSI, DSI Laboratories; UMU, Upstate Medical University; TCH, TexasChildren's Hospital.^(b)NP. Nasopharyngeal swab; NW, nasal wash.^(c)CX, culture in RhMK cell monolayer.^(d)RA, rapid assay.^(e)NT, not tested, RT-PCR was not performed on this specimen becausethe lateral-flow assay was positive at 30 min.^(f)Not tested, RT-PCR was not performed because the quantity ofspecimen available was insufficient.^(g)Repeat RA was also negative. Respiratory syncytial virus was alsodetected on day 4 in Hep-2 monolayer cell culture. The possibility of across-contamination of cell tubes, causing a false-positive cell cultureresult for influenza A virus, cannot be excluded as a possible cause ofdiscrepant results with sample 171.

The immunoassay was both highly sensitive and specific in detecting anddifferentiating influenza A and B viruses in respiratory specimenscollected from patients in three different geographic locations during arecent influenza season in the United States. The results were favorablewhen statistically generalized to a larger population, making the assayuseful for screening large numbers of individuals. Many previous studieson the performance of rapid assays for detecting influenza virus werelimited because they were conducted at a single center or during aninfluenza season where only type A predominated. In addition, in the fewstudies in which influenza B virus circulated, significant rates offalse-negative tests have been observed. Dual infections with one typeof influenza virus and another virus were observed in this study,providing a reminder that a positive rapid test for influenza A or Bvirus does not eliminate the possibility that the patient may becoinfected with another virus that may be contributing to theirsymptoms.

The current availability of at least seven different test kits for therapid detection of influenza virus in clinical samples not only enhancesindividual patient care, but also may help control the spread ofinfluenza if infected individuals are accurately diagnosed and treatedpromptly and if outbreaks are identified early and controlled by timelyimmunization practices. However, healthcare workers caring directly forpatients, as well as laboratory directors and public health officials,should be aware of the performance characteristics, availability, cost,and reimbursement issues associated with each rapid test and choose thebest one for their specific needs and, once implemented, monitor theperformance of the test in their particular setting.

In addition to the above examples, other target analytes, includingantigens indicative of infectious disease, hormones, antibodies,receptors, and the like, present suitable analytes for detection usingdextran-polystreptavidin conjugates as capture reagents in conjunctionwith appropriate biotinylated and labeled conjugates to detect targetanalytes with suitable specificity and sensitivity.

All cited publications or references are expressly incorporated byreference herein in their entirety.

Other variations or embodiments of the invention will also be apparentto one skilled in the art from the above description, figures, andexamples. Thus, the foregoing embodiments are not to be construed aslimiting the scope of the invention.

1. An immunoassay method comprising use of a dextran-polystreptavidinconjugate immobilized at a discrete site on a porous carrier material,the dextran-polystreptavidin conjugate deposited at the discrete siteand then incubated under conditions sufficient to immobilize thedextran-polystreptavidin conjugate at the discrete site.
 2. The methodof claim 1 wherein immobilization occurs in the absence of a vacuum. 3.The method of claim 1 wherein immobilization occurs in the absence oflatex particles.
 4. The method of claim 1 wherein incubation is forabout 10 h to about 15 h at about 60° C., or for about 7 days to about14 days at about 20° C.
 5. The method of claim 1 used in a lateral flowimmunoassay.
 6. An immunoassay amplification system comprising a porouscarrier containing a dextran-polystreptavidin conjugate immobilized at atest site for a target analyte, the target analyte comprising a targetantigen or a target antibody, at least one biotinylated antibody orantigen, or fragment thereof, for a respective target antigen orantibody, at least one labeled antibody or antigen, or fragment thereof,for the respective target antigen or antibody, thepolystreptavidin-dextran conjugate capturing the biotinylated antibody,antigen, or fragment thereof at the test site, the biotinylatedantibody, antigen, or fragment thereof forming a sandwich at the testsite with target analyte, if present, and the labeled antibody, antigen,or fragment thereof.
 7. The system of claim 6 wherein the target analyteis at least one of influenza A antigen or influenza B antigen.
 8. Thesystem of claim 6 wherein the target analyte is at least one of aninfectious disease antigen, an antibody, a hormone, or a receptor.
 9. Asolid phase immunoassay amplification method comprisingdextran-polystreptavidin deposited at a test site on a porous carrierunder conditions sufficient to immobilize the dextran-polystreptavidinat the test site and thus amplify the amount of a target analyte boundto a labeled antibody at the test site in a sandwich immunoassay. 10.The method of claim 9 wherein the method sandwiches the target antigenat the test site with a biotinylated antibody bound to thepolystreptavidin at the test site and a labeled antibody to the targetantigen.
 11. The method of claim 9 wherein the biotinylated and labeledantibodies are the same or different.
 12. The method of claim 9 whereinthe label is at least one of an enzyme, an enzyme fragment, a metal sol,a latex particle, a fluorescent molecule, or a dye.
 13. The method ofclaim 9 wherein the biotinylated antibody is provided at a discreteunoccupied site on the porous carrier.
 14. The method of claim 9 whereinthe biotinylated antibody is provided on a discrete porous carrier. 15.A solid phase immunoassay comprising a first porous carrier comprising adextran-polystreptavidin conjugate immobilized at a test site, a secondporous carrier upstream of and in wicking flow with the first porouscarrier, the second porous carrier comprising a sample receiving areaand a conjugate site, the conjugate site comprising a mobilizablebiotinylated antibody, and a labeled antibody, the antibodies directedagainst a target antigen.
 16. The immunoassay of claim 15 furthercomprising a backing layer.
 17. The immunoassay of claim 15 furthercomprising an absorbent pad downstream of said test site and optionalcontrol site.
 18. The immunoassay of claim 15 in a single housing.